Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation.

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived ß-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Sphingobium yanoikuyae Bacteremia, Japan.

We report a case of Sphingobium yanoikuyae bacteremia in an 89-year-old patient in Japan. No standard antimicrobial regimen has been established for S. yanoikuyae infections. However, ceftriaxone and ceftazidime treatments were effective in this case. Increased antimicrobial susceptibility data are needed to establish appropriate treatments for S. yanoikuyae.

Comprehensive review on Haloalkane dehalogenase (LinB): a ß-hexachlorocyclohexane (HCH) degrading enzyme.

Haloalkane dehalogenase, LinB, is a member of the α/ß hydrolase family of enzymes. It has a wide range of halogenated substrates, but, has been mostly studied in context of degradation of hexachlorocyclohexane (HCH) isomers, especially ß-HCH (5-12% of total HCH isomers), which is the most recalcitrant and persistent among all the HCH isomers. LinB was identified to directly act on ß-HCH in a one or two step transformation which decreases its toxicity manifold. Thereafter, many studies focused on LinB including its structure determination using X-ray crystallographic studies, structure comparison with other haloalkane dehalogenases, substrate specificity and kinetic studies, protein engineering and site-directed mutagenesis studies in search of better catalytic activity of the enzyme. LinB was mainly identified and characterized in bacteria belonging to sphingomonads. Detailed sequence comparison of LinB from different sphingomonads further revealed the residues critical for its activity and ability to catalyze either one or two step transformation of ß-HCH. Association of LinB with IS6100 elements is also being discussed in detail in sphingomonads. In this review, we summarized vigorous efforts done by different research groups on LinB for developing better bioremediation strategies against HCH contamination. Also, kinetic studies, protein engineering and site directed mutagenesis studies discussed here forms the basis of further exploration of LinB's role as an efficient enzyme in bioremediation projects.

Biodegradation of DEP, DIBP, and BBP by a psychrotolerant Sphingobium yanoikuyae strain P4: Degradation potentiality and mechanism study.

Phthalic acid esters (PAEs) are human made chemicals widely used as plasticizers to enhance the flexibility of plastic products. Due to the lack of chemical bonding between phthalates and plastics, these materials can easily enter the environment. Deleterious effects caused by this chemo-pollutant have drawn the attention of the scientific community to remediate them from different ecosystem. In this context, many bacterial strains have been reported across different habitats and Sphingobium yanoikuyae strain P4 is among the few psychrotolerant bacterial species reported to biodegrade simple and complex phthalates. In the present study, biodegradation of three structurally different PAEs viz., diethyl phthalate (DEP), di-isobutyl phthalate (DIBP), and butyl benzyl phthalate (BBP) have been investigated by the strain P4. Quantitative analyses through High-performance liquid chromatography (HPLC) revealed that the bacterium completely degraded 1 g/L of DEP, DIBP, and BBP supplemented individually in minimal media pH 7.0 within 72, 54, and 120 h of incubation, respectively, at 28 °C and under shake culture condition (180 rpm). In addition, the strain could grow in minimal media supplemented individually with up to 3 g/L of DEP and 10.0 g/L of DIBP and BBP at 28 °C and pH 7.0. The strain also could grow in metabolites resulting from biodegradation of DEP, DIBP, and BBP, viz. n-butanol, isobutanol, butyric acid, ethanol, benzyl alcohol, benzoic acid, phthalic acid, and protocatechuic acid. Furthermore, phthalic acid and protocatechuic acid were also detected as degradation pathway metabolites of DEP and DIBP by HPLC, which gave an initial idea about the biodegradation pathway(s) of these phthalates.

Sphingobium cyanobacteriorum sp. nov., isolated from fresh water.

A novel Gram-stain-negative, yellow-pigmented, short rod-shaped bacterial strain, HBC34T, was isolated from a freshwater sample collected from Daechung Reservoir, Republic of Korea. The results of 16S rRNA gene sequence analysis indicated that HBC34T was affiliated with the genus Sphingobium and shared the highest sequence similarity to the type strains of Sphingobium vermicomposti (98.01 %), Sphingobium psychrophilum (97.87 %) and Sphingobium rhizovicinum (97.59 %). The average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values between HBC34T and species of the genus Sphingobium with validly published names were below 84.01 and 28.1 %, respectively. These values were lower than the accepted species-delineation thresholds, supporting its recognition as representing a novel species of the genus Sphingobium. The major fatty acids (>10 % of the total fatty acids) were identified as summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The main polar lipids were phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, two phospholipids and two unidentified polar lipids. The respiratory quinone was Q-10. The genomic DNA G+C content of HBC34T was 64.04 %. The polyphasic evidence supports the classification of HBC34T as the type strain of a novel species of the genus Sphingobium, for which the name Sphingobium cyanobacteriorum sp. nov is proposed. The type strain is HBC34T (= KCTC 8002T= LMG 33140T).

Bacterial community assessment of drinking water and downstream distribution systems in highland localities of Ecuador.

Bacterial communities in drinking water provide a gauge to measure quality and confer insights into public health. In contrast to urban systems, water treatment in rural areas is not adequately monitored and could become a health risk. We performed 16S rRNA amplicon sequencing to analyze the microbiome present in the water treatment plants at two rural communities, one city, and the downstream water for human consumption in schools and reservoirs in the Andean highlands of Ecuador. We tested the effect of water treatment on the diversity and composition of bacterial communities. A set of physicochemical variables in the sampled water was evaluated and correlated with the structure of the observed bacterial communities. Predominant bacteria in the analyzed communities belonged to Proteobacteria and Actinobacteria. The Sphingobium genus, a chlorine resistance group, was particularly abundant. Of health concern in drinking water reservoirs were Fusobacteriaceae, Lachnospiraceae, and Ruminococcaceae; these families are associated with human and poultry fecal contamination. We propose the latter families as relevant biomarkers for establishing local standards for the monitoring of potable water systems in highlands of Ecuador. Our assessment of bacterial community composition in water systems in the Ecuadorian highlands provides a technical background to inform management decisions.

Stereoinversion via Alcohol Dehydrogenases Enables Complete Catabolism of ß-1-Type Lignin-Derived Aromatic Isomers.

Sphingobium sp. strain SYK-6 is an efficient aromatic catabolic bacterium that can consume all four stereoisomers of 1,2-diguaiacylpropane-1,3-diol (DGPD), which is a ring-opened ß-1-type dimer. Recently, LdpA-mediated catabolism of erythro-DGPD was reported in SYK-6, but the catabolic pathway for threo-DGPD was as yet unknown. Here, we elucidated the catabolism of threo-DGPD, which proceeds through conversion to erythro-DGPD. When threo-DGPD was incubated with SYK-6, the Cα hydroxy groups of threo-DGPD (DGPD I and II) were initially oxidized to produce the Cα carbonyl form (DGPD-keto I and II). This initial oxidation step is catalyzed by Cα-dehydrogenases, which belong to the short-chain dehydrogenase/reductase (SDR) family and are involved in the catabolism of ß-O-4-type dimers. Analysis of seven candidate genes revealed that NAD+-dependent LigD and LigL are mainly involved in the conversion of DGPD I and II, respectively. Next, we found that DGPD-keto I and II were reduced to erythro-DGPD (DGPD III and IV) in the presence of NADPH. Genes involved in this reduction were sought from Cα-dehydrogenase and ldpA-neighboring SDR genes. The gene products of SLG_12690 (ldpC) and SLG_12640 (ldpB) catalyzed the NADPH-dependent conversion of DGPD-keto I to DGPD III and DGPD-keto II to DGPD IV, respectively. Mutational analysis further indicated that ldpC and ldpB are predominantly involved in the reduction of DGPD-keto. Together, these results demonstrate that SYK-6 harbors a comprehensive catabolic enzyme system to utilize all four ß-1-type stereoisomers through successive oxidation and reduction reactions of the Cα hydroxy group of threo-DGPD with a net stereoinversion using multiple dehydrogenases. IMPORTANCE In many catalytic depolymerization processes of lignin polymers, aryl-ether bonds are selectively cleaved, leaving carbon-carbon bonds between aromatic units intact, including dimers and oligomers with ß-1 linkages. Therefore, elucidating the catabolic system of ß-1-type lignin-derived compounds will aid in the establishment of biological funneling of heterologous lignin-derived aromatic compounds to value-added products. Here, we found that threo-DGPD was converted by successive stereoselective oxidation and reduction at the Cα position by multiple alcohol dehydrogenases to erythro-DGPD, which is further catabolized. This system is very similar to that developed to obtain enantiopure alcohols from racemic alcohols by artificially combining two enantiocomplementary alcohol dehydrogenases. The results presented here demonstrate that SYK-6 has evolved to catabolize all four stereoisomers of DGPD by incorporating this stereoinversion system into its native ß-1-type dimer catabolic system.

Sphingobium lignivorans sp. nov., isolated from river sediment downstream of a paper mill.

A bacterial isolate, B1D3AT, was isolated from river sediment collected from the Hiwassee River near Calhoun, TN, by enrichment culturing with a model 5-5' lignin dimer, dehydrodivanillate, as its sole carbon source. B1D3AT was also shown to utilize several model lignin-derived monomers and dimers as sole carbon sources in a variety of minimal media. Cells were Gram-stain-negative, aerobic, motile, rod-shaped and formed yellow/cream-coloured colonies on rich agar. Optimal growth occurred at 30 °C, pH 7-8, and in the absence of NaCl. The major fatty acids of B1D3AT were C18 : 1 ω7c and C17 : 1 ω6c. The predominant hydroxy fatty acids were C14 : 0 2-OH and C15 : 0 2-OH. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethanolamine and sphingoglycolipid. B1D3AT contained spermidine as the only major polyamine. The major isoprenoid quinone was Q-10 with minor amounts of Q-9 and Q-11. The genomic DNA G+C content of B1D3AT was 65.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 49 core, universal genes defined by Clusters of Orthologous Groups gene families indicated that B1D3AT was a member of the genus Sphingobium. B1D3AT was most closely related to Sphingobium sp. SYK-6, with a 100 % 16S rRNA gene sequence similarity. B1D3AT showed 78.1-89.9 % average nucleotide identity and 19.5-22.2% digital DNA-DNA hybridization identity with other type strains from the genus Sphingobium. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain B1D3AT should be classified as representing a novel species of the genus Sphingobium, for which the name Sphingobium lignivorans sp. nov. is proposed. The type strain is strain B1D3AT (ATCC TSD-279T=DSM 111877T).

Sphingobium nicotianae sp. nov., isolated from tobacco soil.

Bacterial strain H33T was isolated from tobacco plant soil and was characterized using a polyphasic taxonomy approach. Strain H33T was a Gram-stain-negative, rod-shaped, non-motile and strictly aerobic bacterium. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of the up-to-date bacterial core gene set (92 protein clusters) indicated that H33T belongs to the genus Sphingobium. Strain H33T showed the highest 16S rRNA gene sequence similarity to Sphingobium xanthum NL9T (97.2%) and showed 72.3-80.6 % average nucleotide identity and 19.7-29.2 % digital DNA-DNA hybridization identity with the strains of other species of the genus Sphingobium. Strain H33T grew optimally at 30°C, pH 7 and could tolerate 0.5 % (w/v) NaCl. The isoprenoid quinones were ubiquinone-9 (64.1%) and ubiquinone-10 (35.9%). Spermidine was the major polyamine. The major fatty acids of H33T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The polar lipid profile consisted of a mixture of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, sphingoglycolipid, two unidentified lipids, two unidentified glycolipids, two unidentified aminoglycolipids and an unidentified phospholipid. The genomic DNA G+C content of H33T was 64.9 mol%. Based on the phylogenetic and phenotypic data, H33T was considered a representative of a novel species in the genus Sphingobium. We propose the name Sphingobium nicotianae sp. nov., with H33T (=CCTCC AB 2022073T=LMG 32569T) as the type strain.

Sphingobium agri sp. nov., isolated from rhizospheric soil of eggplant.

A Gram-stain-negative, aerobic, short rod-shaped and motile bacterial strain, designated MAH-33T, was isolated from rhizospheric soil of eggplant. The colonies were observed to be yellow-coloured, smooth, spherical and 0.1-0.3 mm in diameter when grown on TSA agar medium for 2 days. Strain MAH-33T was found to be able to grow at 10-40 °C, at pH 5.0-10.0 and at 0-3.0 % NaCl (w/v). The strain was found to be positive for both oxidase and catalase tests. The strain was positive for hydrolysis of tyrosine and aesculin. According to the 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Sphingobium and to be closely related to Sphingobium quisquiliarum P25T (98.4 % similarity), Sphingobium mellinum WI4T (97.8 %), Sphingobium fuliginis TKPT (97.3 %) and Sphingobium herbicidovorans NBRC 16415T (96.9 %). The novel strain MAH-33T has a draft genome size of 3 908 768 bp (28 contigs), annotated with 3689 protein-coding genes, 45 tRNA and three rRNA genes. The average nucleotide identity and digital DNA-DNA hybridization values between strain MAH-33T and closely related type strains were in the range of 79.8-81.6 % and 23.2-24.5 %, respectively. The genomic DNA G+C content was determined to be 62.2 %. The predominant isoprenoid quinone was ubiquinone 10. The major fatty acids were identified as C16 : 0 and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The polar lipids identified in strain MAH-33T were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, sphingoglycolipid, phosphatidylcholine; one unknown phospholipid and one unknown lipid. On the basis of digital DNA-DNA hybridization, ANI value, genotypic analysis, chemotaxonomic and physiological data, strain MAH-33T represents a novel species within the genus Sphingobium, for which the name Sphingobium agri sp. nov. is proposed, with MAH-33T (=KACC 19973T = CGMCC 1.16609T) as the type strain.

First Study Case of Microbial Biocontrol Agents Isolated from Aquaponics Through the Mining of High-Throughput Sequencing Data to Control Pythium aphanidermatum on Lettuce.

Aquaponics is defined as a sustainable and integrated system that combines fish aquaculture and hydroponic plant production in the same recirculated water loop. A recent study using high-throughput sequencing (HTS) technologies highlighted that microbial communities from an aquaponic system could control one of the most problematic pathogens in soilless lettuce culture, namely, Pythium aphanidermatum. Therefore, this study aims at isolating the microorganisms responsible for this biocontrol action. Based on the most promising genera identified by HTS, an innovative strategy for isolating and testing original biocontrol agents from aquaponic water was designed to control P. aphanidermatum. Eighty-two bacterial strains and 18 fungal strains were isolated, identified by Sanger sequencing, and screened in vivo to control damping-off of lettuce seeds caused by P. aphanidermatum. Out of these 100 isolates, the eight most efficacious ones were selected and further tested individually to control root rot disease caused by the same pathogen at a later stage of lettuce growth. Strains SHb30 (Sphingobium xenophagum), G2 (Aspergillus flavus), and Chito13 (Mycolicibacterium fortuitum) decreased seed damping-off at a better rate than a propamocarb fungicide and a Pseudomonas chlororaphis registered biocontrol agent did. In root rot bioassays, lettuce mortality was prevented by applying strains G2 and Chito13, which were at least as efficacious as the fungicide or biopesticide controls. Lettuce disease symptoms and mortality were eradicated by strain SHb30 in the first bioassay, but not in the second one. These results show that aquaponic systems are promising sources of original biocontrol agents, and that HTS-guided strategies could represent interesting approaches to identify new biocontrol agents.

Nondesulfurizing benzothiophene biotransformation to hetero and homodimeric ortho-substituted diaryl disulfides by the model PAH-degrading Sphingobium barthaii.

Understanding the biotransformation mechanisms of toxic sulfur-containing polycyclic aromatic hydrocarbon (PASH) pollutants such as benzothiophene (BT) is useful for predicting their environmental fates. In the natural environment, nondesulfurizing hydrocarbon-degrading bacteria are major active contributors to PASH biodegradation at petroleum-contaminated sites; however, BT biotransformation pathways by this group of bacteria are less explored when compared to desulfurizing organisms. When a model nondesulfurizing polycyclic aromatic hydrocarbon-degrading soil bacterium, Sphingobium barthaii KK22, was investigated for its ability to cometabolically biotransform BT by quantitative and qualitative methods, BT was depleted from culture media but was biotransformed into mostly high molar mass (HMM) hetero and homodimeric ortho-substituted diaryl disulfides (diaryl disulfanes). HMM diaryl disulfides have not been reported as biotransformation products of BT. Chemical structures were proposed for the diaryl disulfides by comprehensive mass spectrometry analyses of the chromatographically separated products and were supported by the identification of transient upstream BT biotransformation products, which included benzenethiols. Thiophenic acid products were also identified, and pathways that described BT biotransformation and novel HMM diaryl disulfide formation were constructed. This work shows that nondesulfurizing hydrocarbon-degrading organisms produce HMM diaryl disulfides from low molar mass polyaromatic sulfur heterocycles, and this may be taken into consideration when predicting the environmental fates of BT pollutants.

Phytoremediation of PAH compounds by microbial communities in sodic soil.

The PAH degrading microbial consortium was collected from sodic soil of the nursery of Guru Jambheshwar University of Science and Technology, Hisar, Haryana (India). And the soil was artificially amended with phenanthrene and naphthalene to isolate the PAHs degrading microbial consortium. The diversity of microbial consortium was analyzed using the NGS (Next Generation Sequencing) based metagenomic approach. The result of diversity analysis showed species Tepidanaerobacter syntrophicus, Sphingomonas oliophenolica, Arthrobacter psychrochitinipnius, Bifidobacterium bombi, Nocardiodies islandensis, Rhodovibrio sodomensis, Thiorhodococus pfennigii, Aeromicrobium ponti, Steroidobacter dentrificans, Actinomaduria maheshkhaliensis, Dactylosporangium maewongense, Pelotomaculum isophthalicicum, and Nocardioides islandensis were present in the consortium. Moreover, Sphingomonas, Arthrobacter, Sphingobium, Azospirillium, Thirohodococcus, and Pelotomaculum were the prominent pollutant degrader genera in the microbial consortium. Since the bioremediation of these pollutants occurs with a significant reduction in toxicity, the study's perspective is to use this type of consortium for bioremediation of specifically contaminated soil.

The present work's novelty was to find the helpful microbial consortium for the bioremediation of toxic compounds such as naphthalene and phenanthrene. In this study, the degradation of such compounds was done by the various communities of genera like Bifidobacterium, Conexibacter, Sterodobacter, Rhodovibrio, Arthrobacter, Actinomadura, and Euzebya, which are rarely described in the earlier researches. Therefore, this study will enhance the quality of future research.

Comparative genomics of the plant-growth promoting bacterium Sphingobium sp. strain AEW4 isolated from the rhizosphere of the beachgrass Ammophila breviligulata.

BACKGROUND: The genus Sphingobium within the class Alpha-proteobacteria contains a small number of plant-growth promoting rhizobacteria (PGPR), although it is mostly comprised of organisms that play an important role in biodegradation and bioremediation in sediments and sandy soils. A Sphingobium sp. isolate was obtained from the rhizosphere of the beachgrass Ammophila breviligulata with a variety of plant growth-promoting properties and designated as Sphingobium sp. strain AEW4. RESULTS: Analysis of the 16S rRNA gene as well as full genome nucleotide and amino acid identities revealed that this isolate is most similar to Sphingobium xenophagum and Sphingobium hydrophobicum. Comparative genomics analyses indicate that the genome of strain AEW4 contains unique features that explain its relationship with a plant host as a PGPR, including pathways involved in monosaccharide utilization, fermentation pathways, iron sequestration, and resistance to osmotic stress. Many of these unique features are not broadly distributed across the genus. In addition, pathways involved in the metabolism of salicylate and catechol, phenyl acetate degradation, and DNA repair were also identified in this organism but not in most closely related organisms. CONCLUSION: The genome of Sphingobium sp. strain AEW4 contains a number of distinctive features that are crucial to explain its role as a plant-growth promoting rhizobacterium, and comparative genomics analyses support its classification as a relevant Sphingobium strain involved in plant growth promotion of beachgrass and other plants.

Guiding stars to the field of dreams: Metabolically engineered pathways and microbial platforms for a sustainable lignin-based industry.

Lignin is an important structural component of terrestrial plants and is readily generated during biomass fractionation in lignocellulose processing facilities. Due to lacking alternatives the majority of technical lignins is industrially simply burned into heat and energy. However, considering its vast abundance and a chemically interesting richness in aromatics, lignin is presently regarded both as the most under-utilized and promising feedstock for value-added applications. Notably, microbes have evolved powerful enzymes and pathways that break down lignin and metabolize its various aromatic components. This natural pathway atlas meanwhile serves as a guiding star for metabolic engineers to breed designed cell factories and efficiently upgrade this global waste stream. The metabolism of aromatic compounds, in combination with success stories from systems metabolic engineering, as reviewed here, promises a sustainable product portfolio from lignin, comprising bulk and specialty chemicals, biomaterials, and fuels.

The Catabolic System of Acetovanillone and Acetosyringone in Sphingobium sp. Strain SYK-6 Useful for Upgrading Aromatic Compounds Obtained through Chemical Lignin Depolymerization.

Acetovanillone is a major aromatic monomer produced in oxidative/base-catalyzed lignin depolymerization. However, the production of chemical products from acetovanillone has not been explored due to the lack of information on the microbial acetovanillone catabolic system. Here, the acvABCDEF genes were identified as specifically induced genes during the growth of Sphingobium sp. strain SYK-6 cells with acetovanillone and these genes were essential for SYK-6 growth on acetovanillone and acetosyringone (a syringyl-type acetophenone derivative). AcvAB and AcvF produced in Escherichia coli phosphorylated acetovanillone/acetosyringone and dephosphorylated the phosphorylated acetovanillone/acetosyringone, respectively. AcvCDE produced in Sphingobium japonicum UT26S carboxylated the reaction products generated from acetovanillone/acetosyringone by AcvAB and AcvF into vanilloyl acetic acid/3-(4-hydroxy-3,5-dimethoxyphenyl)-3-oxopropanoic acid. To demonstrate the feasibility of producing cis,cis-muconic acid from acetovanillone, a metabolic modification on a mutant of Pseudomonas sp. strain NGC7 that accumulates cis,cis-muconic acid from catechol was performed. The resulting strain expressing vceA and vceB required for converting vanilloyl acetic acid to vanillic acid and aroY encoding protocatechuic acid decarboxylase in addition to acvABCDEF successfully converted 1.2 mM acetovanillone to approximately equimolar cis,cis-muconic acid. Our results are expected to help improve the yield and purity of value-added chemical production from lignin through biological funneling. IMPORTANCE In the alkaline oxidation of lignin, aromatic aldehydes (vanillin, syringaldehyde, and p-hydroxybenzaldehyde), aromatic acids (vanillic acid, syringic acid, and p-hydroxybenzoic acid), and acetophenone-related compounds (acetovanillone, acetosyringone, and 4'-hydroxyacetophenone) are produced as major aromatic monomers. Also, base-catalyzed depolymerization of guaiacyl lignin resulted in vanillin, vanillic acid, guaiacol, and acetovanillone as primary aromatic monomers. To date, microbial catabolic systems of vanillin, vanillic acid, and guaiacol have been well characterized, and the production of value-added chemicals from them has also been explored. However, due to the lack of information on the microbial acetovanillone and acetosyringone catabolic system, chemical production from acetovanillone and acetosyringone has not been achieved. This study elucidated the acetovanillone/acetosyringone catabolic system and demonstrates the potential of using these genes for the production of value-added chemicals from these compounds.

Developing Rhodococcus opacus and Sphingobium sp. coculture systems for valorization of lignin-derived dimers.

Bioconversion is being regarded as a promising way for lignin valorization because it enables funneling diverse lignin components into single compounds, overcoming the heterogeneity of lignin. Although numerous lignin-derived aromatic monomers have been funneled to target compounds in previous studies, the bioconversion of low-molecular-weight lignin (LMW-lignin) fragments, for example, lignin-derived dimers, has been rarely systematically studied, impeding further conversion of lignin. In this study, coculture systems were designed and developed to funnel multiple lignin-derived dimers to cis, cis-muconate and gallate by combining lignin-derived dimers cleavage bacterium Sphingobium sp. and monomers conversion bacterium Rhodococcus opacus. With the developed coculture systems, ß-O-4 type dimer guaiacylglycerol-ß-guaiacyl ether, 4-O-5 type dimer 4,4'-dihydroxydiphenyl ether, ß-5 type dimer balanophonin, ß-ß type dimer pinoresinol, ß-1 type dimer 1,2-bis(4-hydroxy-3-methoxyphehyl)-1,3-propanediol and 5-5 type dimer 5,5'-dehydrodivanillate were converted to cis, cis-muconate. Additionally, the developed coculture systems also showed potential in conversion of lignin-derived dimers to gallate. The application of alkali lignin for cis, cis-muconate production further demonstrated the effectiveness of the designed coculture systems. Overall, the developed coculture systems are beneficial to lignin biological valorization, and also provide references for the valorization of other bio-resources.

Phenanthrene-degrading Sphingobium xenophagum are widely distributed in the western Pacific Ocean.

Six phenanthrene-degrading bacteria were isolated from surface seawater sampled from the western Pacific Ocean. They were identified as Sphingobium xenophagum (formerly Sphingomonas xenophaga) based on morphological, biochemical, and chemical characteristics, and 16S rRNA sequences. The salinity ranges for the growth of these isolates were broader than those of the seven reported Sphingomonas strains isolated from soil, and the optimum NaCl concentration in the growth medium was higher than that for soil sphingomonads. These isolates also exhibited higher phenanthrene-degrading activity under briny conditions than that of a phenanthrene-degrading Sphingomonas strain isolated from soil. A DNA fragment carrying nah genes, which are encoded on the naphthalene-catabolic plasmid NAH of Pseudomonas putida PpG7, hybridised less strongly with the total DNA of all isolates. Certain genes involved in phenanthrene degradation were also preliminarily characterised in all isolates. This is the first demonstration that S. xenophagum strains, which are able to degrade phenanthrene, are widely distributed in marine environments, and that the growth and phenanthrene-degrading activity of these strains are adapted to briny conditions. The results also suggest that genes for phenanthrene degradation, which are dissimilar to nah genes, were also ubiquitously distributed in marine strains.

Comparative Analysis of Bile-Salt Degradation in Sphingobium sp. Strain Chol11 and Pseudomonas stutzeri Strain Chol1 Reveals Functional Diversity of Proteobacterial Steroid Degradation Enzymes and Suggests a Novel Pathway for Side Chain Degradation.

The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called Δ4,6-variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations of strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase (ACAD) Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the Δ22-ACAD Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts, while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 Δscd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshABChol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only Δ4,6-steroids, while KshABChol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228, led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of proteobacterial enzymes for bile-salt degradation and suggest a novel side chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes a proteobacterial ketosteroid-9α-hydroxylase and shows that this enzyme is able to catalyze side reactions with nonnative substrates.

The Syringate O-Demethylase Gene of Sphingobium sp. Strain SYK-6 Is Regulated by DesX, while Other Vanillate and Syringate Catabolism Genes Are Regulated by DesR.

Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA, ligM, and desB are induced by syringate and vanillate, while those of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here, we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX, located downstream of desA Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicated that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB The ferulate catabolism genes (ferBA), under the control of a MarR-type transcriptional regulator, FerC, are located upstream of desA RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster consists of the ferBA operon and the SLG_25010-desA operon. Promoter assays revealed that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps an 18-bp inverted-repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that the compounds are effector molecules of DesX.IMPORTANCE Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and to elucidate their transcriptional regulation. In this study, we found that the transcription of desA, encoding syringate O-demethylase in SYK-6, is regulated by an IclR-type transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (encoding a MarR transcriptional regulator that controls the transcription of ligM and desB), provide an overall picture of the transcriptional-regulatory systems for syringate and vanillate catabolism in SYK-6.